DYRK protein kinases in Chlamydomonas reinhardtii: Biochemical characterization and physiological study of a DYRK mutant strain
Zusammenfassung der Projektergebnisse
Recently, a mutant strain affected in starch degradation (std1) was isolated in a screen of an insertion mutant library of the model alga Chlamydomonas reinhardtii aiming at identifying new regulatory processes controlling accumulation of reserve compounds. The impaired gene was identified as a dual-specificity tyrosine-phosphorylation-regulated kinase (DYRK) and renamed DYRKP-1 as it belongs to a new subgroup of the DYRK protein family containing only members of the green lineage (DYRKP). During the funding period, a more detailed physiological characterization and a first approach for a biochemical analysis were envisaged, and several important new insights into the function of STD1/DYRKP-1 were obtained. Previously, it was shown that the std1 mutant accumulates much higher starch amounts and maintains higher photosynthetic activity than wild-type and two complemented lines under photoautotrophic nitrogen deprivation. Here it was found that this mutant phenotype is not restricted to nitrogen limitation condition, but also caused, when std1 cells were starved for other macronutrients such as sulfur and phosphorus. Furthermore, deprivation kinetics at low light intensities were performed revealing still high starch accumulation in the mutant. A rapid break-down of starch with an again subsequent increase after repletion of the medium during illumination was observed. Remarkably, the std1 mutant showed a continuous increase in total cellular volume and starch reserves during long-term growth in photoautotrophic condition with high CO2, which was not the case in air or standard mixotrophic conditions. All these results suggest that the STD1/DYRKP-1 protein kinase is part of a signaling pathway responding to general nutrient depletion (N, S, P or during stationary growth phase) and secondly, that the link between cellular energy status and starch accumulation is lost in the mutant. The successful antibody production and immunodetection of STD1/DYRKP-1 in this project constitutes an important tool for a further biochemical characterization. Because transformation and screening of HA-tagged complemented clones and subcellular localization by immunofluorescence were challenging, subcellular fractionation experiments were carried out suggesting that the STD1/DYRKP-1 is localized in the cytosol at normal growth conditions. Furthermore, the time during the funding period allowed us to study lipid accumulation in the std1 mutant. The high biomass, starch and oil accumulating phenotype of this mutant may attract great interest for biotechnological development. The data gathered so far provided promising directions for further studies of altered lipid profiles in the mutant helping to elucidate the DYRKP-1 signaling pathway. Another important achievement was the preparation of samples from cells grown in photobioreactors for a comparative transcriptome analysis by RNA-Seq technology. These data are supposed to provide new insights into the function of STD1/DYRKP-1 and prompt to further investigations in the future.
